Composition for targeting, storing and loading of liposomes

ABSTRACT

The present invention describes a composition consisting of liposomes covalently or non-covalently coupled to the glycoprotein streptavidin. The streptavidin may additionally be coupled to biotinated proteins such as Immunoglobulin G or monoclonal antibodies. 
     The liposomes of the invention may have a transmembrane potential across their membranes, and may be dehydrated. In addition, the composition may contain ionizable bioactive agents such as antineoplastic agents, and may be used in diagnostic assays.

This is a continuation-in-part of copending application Ser. No. 811,037, filed Dec. 18, 1985, now abandoned , which in turn is acontinuation-in-part of copending application Ser. No. 749,161, nowabandoned, filed June 26, 1985, and copending application Ser. No.759,419, filed July 26, 1985.

BACKGROUND OF THE INVENTION

The present invention is directed to liposomes. More particularly, thepresent invention is directed to the covalent and non-covalent couplingof liposomes to proteins for purposes of in vivo targeting, or for usein diagnostics.

Liposomes are completely closed structures composed of lipid bilayermembranes containing an encapsulated aqueous volume. Liposomes maycontain many concentric lipid bilayers separated by aqueous phase(multilamellar vesicles or MLVs), or may be composed of a singlemembrane bilayer (unilamellar vesicles).

Liposome preparation has typically been achieved by the process ofBangham et.al., (1965 J. Mol. Biol., 13: 238-252) whereby lipidssuspended in organic solvent are evaporated under reduced pressure to adry film in a reaction vessel. An appropriate amount of aqueous phase isthen added to the vessel and the mixture agitated, then allowed tostand, essentially undisturbed for a time sufficient for themultilamellar vesicles to form. The aqueous phase entrapped within theliposomes may comprise bioactive agents including, but not limited to,drugs, hormones, proteins, dyes, vitamins, or imaging agents.

The current state of the art is such that liposomes may be reproduciblyprepared using a number of techniques. Liposomes resulting from some ofthese techniques are small unilamellar vesicles (SUVs) Papahadjopoulosand Miller, Biochem. Biophys. Acta, 135, p. 624-638 (1967),reverse-phase evaporation vesicles (REV) U.S. Pat. No. 4,235,871 issuedNov. 25, 1980, stable plurilamellar vesicles (SPLV) U.S. Pat. No.4,522,803 issued June 11, 1985, and large unilamellar vesicles producedby an extrusion technique as described in copending U.S. patentapplication Ser. No. 788,017, filed Oct. 16, 1985, Cullis et.al.,entitled "Extrusion Technique for Producing Unilamellar Vesicles",relevant portions of which are incorporated herein by reference.

One of the primary uses for liposomes is as carriers for a variety ofmaterials, such as, drugs, cosmetics, diagnostic reagents, bioactivecompounds, and the like. Such systems may be designed for bothdiagnostics and in vivo uses. In this regard, the ability to produce anantibody-directed vesicle would be a distinct advantage over similarundirected systems (Gregoriadis, G., Trends Pharmacol Sci, 4, p.304-307, 1983). Useful applications would be in the selective targetingof cytotoxic compounds entrapped in vesicles to circulating tumor cells(Wolff et.al., Biochim Biophys. Acta, 802, p. 259-273 1984), orapplications of these immunoglobulin-associated vesicles in thedevelopment of diagnostic assays. As is well known in the art, liposomesmay be covalently coupled to proteins, antibodies and immunoglobins.Heath et.al. (Biochim. Biophys. Acta., 640, p. 66-81, 1981), describethe covalent attachment of immunoglobulins to liposomes containingglycosphingolipid. Leserman et. al. (Liposome Technology, III, 1984, CRCPress, Inc., CA., p. 29-40; Nature, 288, p. 602-604, 1980) and Martinet. al., (J. Biol. Chem., 257, p. 286-288, 1982) have describedprocedures whereby thiolated IgG or protein A is covalently attached tolipid vesicles, and thiolated antibodies and Fab' fragments are attachedto liposomes, respectively. These protocols and various modifications(Martin et.al, Biochemistry, 20, p. 4229-4238, 1981; and Goundalkaret.al., J. Pharm. Pharmacol. 36, p. 465-466, 1984) represent the mostversatile approaches to coupling. Avidin-coupled and avidin andbiotinyl-coupled phospholid liposomes containing actinomycin D havesuccessfully targeted tumor cells expressing ganglio-N-triosylceramide(Urdal et al., J. Biol. Chem., 255, p. 10509-10516, 1980). Huang et al.(Biochim. Biophys. Acta., 716, p. 140-150, 1982) demonstrate the bindingof mouse monoclonal antibody to the major histocompatibility antigen H-2(K), or goat antibody to the major glycoprotein of Molony LeukemiaVirus, to palmitic acid. These fatty acid modified IgGs wereincorporated into liposomes, and the binding of these liposomes to cellsexpressing the proper antigens characterized. Other in vitro efforts tospecific binding of liposomes coated with specific immunoglobins havebeen performed (Sharkey et al., Fed. Proc., 38, p. 1089, 1979). In stillother coupling studies, Rahman et. al. found that tissue uptake ofliposomes could be altered by attachment of glycolipids to the liposomes(J. Cell Biol., 83, p. 268a, 1979).

One aspect of the present invention is to couple biotinylated proteinssuch as immunoglobulins and antibodies to liposomes withcovalently-attached streptavidin. Methods for this coupling are hereinprovided. The nature of this covalent attachment between streptavidinand the liposomes is a chemical bonding between the streptavidin, andderivatized phosphatidylethanolamine incorporated in the liposomebilayer. In a second aspect of the invention, Applicants provide atwo-step method for the non-covalent coupling of these biotinylatedproteins to biotinylated-phosphatidylethanolamine (PE)-containingliposomes through the same streptavidin linker. This non-covalentattachment of streptavidin and liposomes occurs through a specificassociation between four specific biotin binding sites on streptavidin,and the biotin. These antibody-liposome complexes bind specifically totarget cells as directed by the coupled antibody. Such liposomes may bemade to contain bioactive agents such as drugs.

In accordance with a primary use for liposomes, the entrapment ofantineoplastic agents inside liposomal bilayers has resulted in moreefficacious therapy as compared to direct administration of the drug.(Forsben et al., Cancer Res., 43, p. 546, 1983; and Gabizon et al.,Cancer Res., 42, p. 4734, 1982). A problem with the encapsulation ofantineoplastic drugs is in the fact that many of these drugs have beenfound to be rapidly released from liposomes after encapsulation. This isan especially undesirable effect, in view of the fact that toxicity ofthese agents can be significantly reduced through liposome encapsulationas compared to direct administration. See, for example, Forssen et al.Cancer Res. 43, 546 (1983) and Rahman et al. Cancer Res., 42, 1817(1982). Clearly, a method whereby drug could be loaded into preformedliposomes would be advantageous. To achieve this object, the invention,in accordance with one of its aspects, provides a method for loadingliposomes with ionizable antineoplastic agents wherein a transmembranepotential is created across the walls of the liposomes and theantineoplastic agent is loaded into the liposomes by means of thetransmembrane potential. See also U.S. patent application Ser. No.749,161, Bally et al. entitled "Encapsulation of Antineoplastic Agentsin Liposomes", filed June 26, 1985, relevant portions of which areincorporated herein by reference.

In accordance with these needs, a liposome composition is presentedwhich describes the use of protein-coupled liposomes which may be storedstably for an indefinite period, in a dehydrated state, with loading ofthe liposomes on an "as needed" basis.

SUMMARY OF THE INVENTION

We have prepared a liposome composition whereby the glycoproteinstreptavidin is coupled to liposomes for purposes of liposome targeting.The streptavidin may in turn couple biotinated proteins such asImmunoglobulin G or monoclonal antibodies and be loaded with a varietyof bioactive agents, depending on use. Such agents may be theantineoplastic agents such as daunorubicin, doxorubicin, andvinblastine.

The liposomes are preferably prepared in such a way as to create atransmembrane potential across their lamellae in response to aconcentration gradient. This concentration gradient may be created byeither Na⁺ /K⁺ potential or pH (H⁺). The difference in internal versusexternal potential is the mechanism which drives the loading of theliposomes with ionizable bioactive agents; delayed loading of preformedliposomes will occur in response to the transmembrane potential. Theseliposomes may be dehydrated in the presence of one or more protectingsugars such as the disaccharides trehalose and sucrose, stored in theirdehydrated condition, and subsequently rehydrated with retention of theion gradient and associated ability to accumulate the bioactive agent.Such bioactive agents may be those used as in vivo pharmaceuticalpreparations, such as antineoplastic agents including doxorubicin. Thesepreparations may be administered to a subject for treatment of disease.Alternatively, the coupled liposome preparations may be used indiagnostic assays. Methods are provided for the preparation of liposomeseither covalently or non-covalently coupled to streptavidin, which inturn are complexed with biotinylated proteins such as IgG or monoclonalantibodies. In the case of non-covalent binding of liposomes tostreptavidin, the liposomes comprise biotinylatedPhosphatidylethanolamine. Such liposomes are incubated with, forexample, about 10-fold molar excess streptavidin to biotinylatedphosphatidylethanolamine (PE), to complete the coupling reaction. Theliposomes may be large unilamellar vesicles, and may also comprise eggPhosphatidylcholine (EPC).

In preparations containing EPC and biotinylated PE, the latter is in anabout 0.1 to 0.5% mole ratio with the EPC, preferably an about 0.1% moleratio.

Compositions of protein-streptavidin-biotinylated PE liposomes whereinthe protein is a monoclonal antibody or Immunoglobulin G are claimed.These liposomes may also comprise a bioactive agent. They may be used invivo as a pharmaceutical preparation in a subject or alternatively in invitro diagnostic assays by contacting a sample with the composition.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph showing coupling of anti-rat erythrocyte IgG to EPCLUVETs (labeled with ³ H-DPPC) containing PDP-PE (closed triangle) andMPB-PE (closed circle) as a function of cholesterol content.

FIG. 2 is a graph characterizing the covalent coupling reaction of IgGto vesicles with regards to time course, of (A), MPB-PE concentration(B) and IgG concentration (C).

FIG. 3 is a graph showing the influence of reaction pH on covalentcoupling of anti-human erythrocyte IgG (closed circle) and streptavidin(closed triangle) to EPC/Chol (50:45) vesicles containing 5 mol %MPB-PE.

FIG. 4 is a graph showing the efficiency of covalent coupling ofanti-human erythrocyte IgG to vesicles of variable size.

FIG. 5 is a graph showing the elution profile for biotinated anti-humanerythrocyte IgG to vesicles which have covalently coupled streptavidin.

FIG. 6 is a graph showing the accumulation of adriamycin intocovalently-coupled streptavidin-vesicles ("avisomes") which wereprepared with a transmembrane pH gradient. Vesicles composed of EPC(closed circle) or EPC/Chol (closed triangle).

FIG. 7 is a graph showing the non-covalent coupling of streptavidin toLUVs containing biotinylated phosphatidylethanolamine.

DETAILED DESCRIPTION OF THE INVENTION

As described above, the present invention describes a liposomecomposition that results from the coupling of the liposomes tostreptavidin. In addition, the composition can be dehydrated andrehydrated. The liposome portions can be loaded with a chosen bioactiveagent by potential difference of ions across the bilayer membranesduring the rehydration step or subsequently thereto. Alternatively, thebioactive agent may be added to the liposomes prior to dehydration. Thestreptavidin-coupled liposomes can be coupled to proteins such asImmunoglobulin G or monoclonal antibodies which have been biotinated bycoupling to biotin. Quite surprising is the observed stability of thestreptavidin-liposome-biotinated protein complex which makesstreptavidin an attractive coupler between the liposomes and thetargeting proteins. The proteins bound to the liposomes aid in targetingthe liposomes and their contents to a specific site in the body.

In one embodiment of the present invention, liposomes are formed usingthe LUVET apparatus described in copending U.S. patent applicationentitled "Extrusion Technique for Producing Unilamellar Vesicles", Ser.No. 622,690, filed June 20, 1984, relevant portions of which areincorporated herein by reference, and covalently coupled to streptavidinusing a modified technique of Leserman et al., (Liposome Technology,III, 1984, CRC Press, Inc., N.Y., p. 29-40). Liposomes are formed with atransmembrane potential i.e. Na⁺ /K⁺ gradient or H⁺ potential differenceacross the bilayers, see copending U.S. patent application, Ser. No.749,161, Bally et al., entitled "Encapsulation of Antineoplastic Agentsin Liposomes", filed June 26, 1985, relevant portions of which areincorporated herein by reference; this potential difference effected bythe ionic concentrations of the internal versus the external media ofthe liposome. Liposomes are then dehydrated either in the presence orabsence of sugars such as trehalose, and may be stored in this state forindefinite periods of time; see copending U.S. patent application, Ser.No. 759,419, Janoff et al., entitled "Dehydrated Liposomes," filed July26, 1985, relevant portions of which are incorporated herein byreference.

In another embodiment of the present invention, biotinylated proteinsare non-covalently coupled to biotinylated PE-containing liposomes viastreptavidin. The non-covalent binding of the streptavidin to theliposomes, the first step, involves incorporation of biotin-PE in theliposomes, followed by a second step of binding the streptavidin to thebiotinylated protein. The proteins are prepared for this binding by theuse of fluorescent derivatizing reagents such as the fluorescent aminereagent fluorescein-isothiocyanate (FITC).

There are four biotin binding sites on the streptavidin, which makesliposomes containing biotin aggregate with streptavidin in an excess ofbiotinylated PE. Thus, the amount of biotinylated PE to incorporate intothe liposomes was titrated in order to prevent this aggregation, whilemaximizing the streptavidin coupling. Values for biotinylated PE mayrange from about 0.05 to 0.5 mole % (with respect to total lipid in theliposome preparation); if the amount of biotin is increased further thanabout 0.5%, complete aggregation and precipitation of liposomes isobserved on addition of streptavidin. This aggregation phenomenon may beexploited in the use of these systems in an aggregation-type diagnosticassay.

The biotinylated antibody is then attached to the streptavidin coatedliposome. These liposomes effectively targeted specifically to theirtarget cells with little non-specific binding.

The liposomes used in the present invention can have a variety ofcompositions and internal contents, and can be in the form ofmultilamellar, unilamellar, or other types of liposomes, or moregenerally, lipid-containing particles, now known or later developed. Forexample, the lipid-containing particles can be in the form of steroidalliposomes, Ser. No. 599,691, now abandoned, alpha-tocopherol containingliposomes, Ser. No. 786,740, now abandoned, stable plurilamellarliposomes (SPLVs), U.S. Pat. No. 4,522,803, issued June 11, 1985,monophasic vesicles (MPVs), U.S. Pat. No. 4,588,578, issued May 13,1986, or lipid matrix carriers (LMC), U.S. Pat. No. 4,610,868, issuedSept. 9, 1986, the pertinent portions of which are incorporated hereinby reference. Within the class of liposomes that may be used in thepresent invention is a preferred subclass of liposomes characterized inhaving solute distribution substantially equal to the solutedistribution environment in which prepared. This subclass may be definedas stable plurilamellar vesicles (SPLV), monophasic vesicles (MPVs), andfrozen and thawed multilamellar vesicles (FATMLVs) as described in"Solute Distributions and Trapping Efficiencies Observed inFreeze-Thawed Multilamellar Vesicles" Mayer et al. Biochimica etBiophysica Acta 817:1983-196 (1985). It is believed that the particularstability of the SPLV type liposomes arises from the low energy stateattendant to solute equilibrium.

Alternatively, techniques used for producing large unilamellar liposomes(LUVs), such as, reverse-phase evaporation, infusion procedures, anddetergent dilution, can be used to produce the liposomes. A review ofthese and other methods for producing liposomes can be found in the textLiposomes, Marc J. Ostro, ed., Marcel Dekker, Inc., New York, 1983,Chapter 1, the pertinent portions of which are incorporated herein byreference.

Compounds which are bioactive agents can be entrapped within theliposomes of the present invention. Such compounds include but are notlimited to antibacterial compounds such as gentamycin, antiviral agentssuch as rifampacin, antifungal compounds such as amphotericin B,anti-parasitic compounds such as antimony derivatives, tumoricidalcompounds such as adriamycin, anti-metabolites, peptides, proteins suchas albumin, toxins such as diptheriatoxin, enzymes such as catalase,polypeptides such as cyclosporin A, hormones such as estrogen, hormoneantagonists, neurotransmitters such as acetylcholine, neurotransmitterantagonists, glycoproteins such as hyaluronic acid, lipoproteins such asalpha-lipoprotein, immunoglobulins such as IgG, immunomodulators such asinterferon or interleuken, vasodilators, dyes such as Arsenazo III,radiolabels such as ¹⁴ C, radio-opaque compounds such as ⁹⁰ Te,fluorescent compounds such as carboxy fluorscein, receptor bindingmolecules such as estrogen receptor protein, anti-inflammatories such asindomethacin, antigalucoma agents such as pilocarpine, mydriaticcompounds, local anesthetics such as lidocaine, narcotics such ascodeine, vitamins such as alpha-tocopherol, nucleic acids such asthymine, polynucleotides such as RNA polymers, psychoactive oranxiolytic agents such as diazepam, mono- di- and polysaccharides, etc.A few of the many specific compounds that can be entrapped arepilocarpine, a polypeptide growth hormone such as human growth hormone,bovin growth hormone and porcine growth hormone, indomethacin, diazepam,alpha-tocopherol itself and tylosin. Antifungal compounds includemiconazole, terconazole, econazole, isoconazole, tioconazole,bifonazole, clotrimazole, ketoconazole, butaconazole, itraconazole,oxiconazole, fenticonazole, nystatin, naftifine, amphotericin B,zinoconazole and ciclopirox olamine, preferably miconazole orterconazole, The entrapment of two or more compounds simultaneously maybe especially desirable where such compounds produce complementary orsynergistic effects. The amounts of drugs administered in liposomes willgenerally be the same as with the free drug; however, the frequency ofdosing may be reduced.

The liposomes of the present invention are preferably dehydrated usingstandard freeze-drying equipment or equivalent apparatus, and, ifdesired, the liposomes and their surrounding medium can be frozen inliquid nitrogen before being dehydrated. Alternatively, the liposomescan also be dehydrated without prior freezing, by simply being placedunder reduced pressure. Dehydration with prior freezing requires thepresence of one or more protective sugars in the preparation. A varietyof sugars can be used, including such sugars as trehalose, maltose,sucrose, glucose, lactose, and dextran. In general, disaccharide sugarshave been found to work better than monsaccharide sugars, with thedisaccharide sugars trehalose and sucrose being most effective.

The one or more sugars are included as part of either the internal orexternal media of the liposomes. Most preferably, the sugars areincluded in both the internal and external media so that they caninteract with both the inside and outside surfaces of the liposomes'membranes. Inclusion in the internal medium is accomplished by addingthe sugar or sugars to the solute which the liposomes are toencapsulate. Since in most cases this solute also forms the bathingmedium for the finished liposomes, inclusion of the sugars in the solutealso makes them part of the external medium. Of course, if an externalmedium other than the original solute is used, e.g., to create atransmembrane potential (see below), the new external medium should alsoinclude one or more of the protective sugars.

In the case of dehydration without prior freezing, if the liposomesbeing dehydrated have multiple lipid layers and if the dehydration iscarried out to an end point where there is sufficient water left in thepreparation so that a substantial portion of the membranes retain theirintegrity upon rehydration, the use of one or more protective sugars maybe omitted. It has been found preferable if the preparation contains atthe end of the dehydration process at least about 2%, and mostpreferably between about 2% and about 5%, of the original water presentin the preparation prior to dehydration.

Once the liposomes have been dehydrated, they can be stored for extendedperiods of time until they are to be used. When the dehydrated liposomesare to be used, rehydration is accomplished by simply adding an aqueoussolution, e.g., distilled water, to the liposomes and allowing them torehydrate.

As discussed above, in accordance with another of its aspects, thepresent invention provides a method for loading liposomes with ionizableantineoplastic agents wherein a transmembrane potential is createdacross the bilayers of the liposomes and the antineoplastic agent isloaded into the liposomes by means of the transmembrane potential. Thetransmembrane potential is generated by creating a concentrationgradient for one or more charged species (e.g., Na⁺, K⁺ and/or H⁺)across the liposome membranes. The concentration gradient is created byproducing liposomes having different internal and external media, i.e.,internal and external media having different concentrations of one ormore charged species.

Specifically, liposomes are prepared which encapsulate a first mediumhaving a first concentration of the one or more charged species. For atypical liposome preparation technique (see discussion above), thisfirst medium will surround the liposomes as they are formed, and thusthe liposomes' original external medium will have the same compositionas the first medium. To create the concentration gradient, the originalexternal medium is replaced by a new external medium having a differentconcentration of the one or more charged species. The replacement of theexternal medium can be accomplished by various techniques, such as, bypassing the liposome preparation through a gel filtration column, e.g.,a Sephadex column, which has been equilibrated with the new medium, orby centrifugation, dialysis, or related techniques.

In accordance with the invention, it has been found that thistransmembrane potential can be used to load ionizable antineoplasticagents into the liposomes. Specifically, once liposomes having aconcentration gradient and thus a transmembrane potential of theappropriate orientation have been prepared, the process of loadingantineoplastic agents into the liposomes reduces to the step of addingthe agent to the external medium. Once added, the transmembranepotential will automatically load the agent into the liposomes.

The transmembrane potential loading method can be used with essentiallyany antineoplastic agent which can exist in a charged state whendissolved in an appropriate aqueous medium (e.g., organic compoundswhich include an amino group which can be protonated). Preferably, theagent should be relatively lipophilic so that it will partition into theliposome membranes. Examples of some of the antineoplastic agents whichcan be loaded into liposomes by this method include doxorubicin,mitomycin, daunorubicin, streptozocin, vinblastine, vincristine,mechlorethamine hydrochloride, melphalan, cyclophosphamide,triethylenethiophosphoramide, carmustine, lomustine, semustine,hydroxyurea, thioguanine, decarbazine, cisplatin, procarbazine, andpharmaceutically acceptable salts and derivatives thereof.

In addition to loading a single antineoplastic agent, the method can beused to load multiple antineoplastic agents, either simultaneously orsequentially. Also, the liposomes into which the ionizableantineoplastic agents are loaded can themselves be pre-loaded with otherantineoplastic agents or other drugs using conventional encapsulationtechniques (e.g., by incorporating the drug in the buffer from which theliposomes are made).

Turning now to the aspects of the invention relating to reducing therate of release of an ionizable antineoplastic agent or other ionizablebiologically-active agent (drug) from liposomes, it has beensurprisingly found that the rate of release can be markedly reduced bycreating a transmembrane potential across the liposome membranes whichis oriented to retain the agent in the liposomes. That is, for an agentwhich is positively charged when ionized, a transmembrane potential iscreated across the liposome membranes which has an inside potentialwhich is negative relative to the outside potential, while for an agentwhich is negatively charged, the opposite orientation is used.

As with the transmembrane loading aspects of the invention, thetransmembrane potentials used to reduce the rate of drug release arecreated by adjusting the concentrations on the inside and outside of theliposomes of a charged species such as Na⁺, K⁺ and/or H⁺. Indeed, if theliposomes have been loaded by means of a transmembrane potentialproduced by such a concentration gradient, simply keeping the liposomesin an external medium which will maintain the original concentrationgradient will produce the desired reduction in the rate of release.Alternatively, if a transmembrane potential has not already been createdacross the liposome membranes, e.g., if the liposomes have been loadedusing a conventional technique, the desired transmembrane potential canbe readily created by changing the composition of the external mediumusing the exchange techniques described above.

Turning next to the aspects of the invention relating to the dehydrationprotocols, two basic approaches are provided: (1) the liposomes can beloaded with antineoplastic agents (e.g., using conventional techniquesor the transmembrane potential loading technique described above),dehydrated for purposes of storage, shipping, and the like, and thenrehydrated at the time of use; or (2) pre-formed liposomes can bedehydrated for storage, etc., and then at or near the time of use, theycan be rehydrated and loaded with an ionizable antineoplastic agentusing the transmembrane potential loading technique described above.

When the dehydrated liposomes are to be used, rehydration isaccomplished by simply adding an aqueous solution, e.g., distilled wateror an appropriate buffer, to the liposomes and allowing them torehydrate. The liposomes can be resuspended into the aqueous solution bygentle swirling of the solution. The rehydration can be performed atroom temperature or at other temperatures appropriate to the compositionof the liposomes and their internal contents.

If the antineoplastic agent which is to be administered was incorporatedinto the liposomes prior to dehydration, and no further compositionchanges are desired, the rehydrated liposomes can be used directly inthe cancer therapy following known procedures for administering liposomeencapsulated drugs.

Alternatively, using the transmembrane potential procedures describedabove, ionizable antineoplastic agents can be incorporated into therehydrated liposomes just prior to administration. In connection withthis approach, the concentration gradient used to generate thetransmembrane potential can be created either before dehydration orafter rehydration using the external medium exchange techniquesdescribed above.

For example,, liposomes having the same internal and external media,i.e., no transmembrane potentials, can be prepared, dehydrated, stored,rehydrated, and then the external medium can be replaced with a newmedium having a composition which will generate transmembranepotentials, and the transmembrane potentials used to load ionizableantineoplastic agents into the liposomes. Alternatively, liposomeshaving internal and external media which will produce transmembranepotentials can be prepared, dehydrated, stored, rehydrated, and thenloaded using the transmembrane potentials.

Liposomes of the present invention may be administered to a subject suchas a mammal including humans. For administration to humans in thetreatment of afflictions, the prescribing physician will ultimatelydetermine the appropriate dose for a given human subject, and this canbe expected to vary according to the age, weight, and response of theindividual as well as the nature and severity of the patient's symptoms.

The mode of administration may determine the sites and cells in theorganism to which the compound will be delivered. For instance, deliveryto a specific site of infection may be most easily accomplished bytopical application (if the infection is external e.g., on areas such aseyes, skin, in ears, or on afflictions such as wounds or burns) or byabsorption through epithelial or mucocutaneous linings (e.g., nasal,oral, vaginal, rectal, gastrointestinal, mucosa, etc.). Such topicalapplication may be in the form of creams or ointments. Theliposome-entrapped materials can be administered alone but willgenerally be administered in admixture with a pharmaceutical carrierselected with regard to the intended route of administration andstandard pharmaceutical practice. They may be injected parenterally, forexample, intravenously, intramuscularly, or subcutaneously. Forparenteral administration, they are best used in the form of a sterileaqueous solution which may contain other solutes, for example, enoughsalts or glucose to make the solution isotonic.

For the oral mode of administration, liposome composition of thisinvention can be used in the form of tablets, capsules, lozenges,troches, powders, syrups, elixirs, aqueous solutions and suspensions,and the like. In the case of tablets, carriers which can be used includelactose, sodium citrate, and salts of phosphoric acid. Variousdisintegrants such as starch, and lubricating agents such as magnesiumstearate, sodium lauryl sulfate and talc, are commonly used in tablets.For oral administration in capsule form, useful diluents are lactose andhigh molecular weight polyethylene glycols. When aqueous suspensions arerequired for oral use, certain sweetening and/or flavoring agents can beadded.

The liposomes of this invention may also be used in diagnostic assays;in this case the amount of the composition used will depend on thesensitivity of the liposome-coupled antibody to the target components inthe sample.

MATERIALS AND METHODS

Egg phospatidylcholine (EPC) was isolated from hen egg yolk employingestablished procedures. Egg phosphatidylethanolamine (EPE) was obtainedfrom EPC utilizing the headgroup exchange capacity of phospholipase D(Kates et al., Methods in Enzymology, 14, Lavenstein, J. ed., 1969,Academic Press, Inc., p. 197-211). Dioleoylphosphatidylcholine (DOPC)and dipalmitoylphosphatidylcholine (DPPC) were obtained from AvantiPolar Lipids. Strepavidin, cholesterol, trehalose, dithiothreitol (DTT),and fluorescein isothiocyanate Celite (Celite FITC) were purchased fromSigma. N-Succinimidyl 3-(2-pyridyldithio) propionate (SPDP),N-succinimidyl 4-(p-Maleimidophenyl)butyrate (SMPB),biotinyl-N-hydroxysuccinimide (BHS) biotin-phosphatidylethanolasine, andN-biotinoyldipalmitoylphosphatidylcholine were obtained from MolecularProbes. Rabbit anti-human red blood cell IgG and Rabbit anti-rat redblood cell IgG were supplied by Cooper Biomedical. Sephadex G-50 fine,Sepharose 4B-CL, and Sepharose C14B were purchased from Pharmacia. [³H]-DPPC and [³ H]-BHS were obtained from NEN, carrier free Na ¹²⁵ I (100mCi/ml) was supplied by Amersham and iodogen was obtained from Pierce.Adriamycin was obtained from B.C. Cancer Control Agency. Biotinylatedanti-rat erythrocyte IgG was obtained from Cappel. All other chemicalswere of analytical grade.

Lipid was estimated by the standard lipid phosphate assay or byincorporation of trace quantities of [³ H-DPPC] introduced in theoriginal lipid film, and later monitored using a Packard Tri-Carb 4000series scintillation counter. FITC was assayed by monitoring thefluorescence at 520 nm using a SLM-Aminco SPF-500C spectrofluotometerwith an excitation wavelength of 495 nm. ¹²⁵ I was measured using aPackard Auto-Gamma 5650 gamma counter. Vesicle size distributions weredetermined by quasi-elastic light scattering (QELS) analysis utilizing aNicomp Model 270 submicron particle sizer operating at 632.8 nm and 5mW.

EXAMPLE 1 Covalent Coupling I. Synthesis ofN-[4-(p-Maleimidophenyl)butyryl] phosphatidylethanolamine (MPB-PE)

MPB-PE was prepared according to the procedure of Martin et al., J.Biol. Chem., 257, 286-288, (1982). EPE (200 mg) was dissolved in 5 ml offreshly distilled anhydrous methanol containing 200 umol of freshlydistilled triethylamine and 100 mg SMBP. The reaction was carried out atroom temperature under nitrogen and its progress followed using thinlayer chromatography (TLC, running solvent:chloroform/methanol/water,65:25:4). Following an 18 hour incubation, 95% of the EPE was convertedto MPB-PE. Methanol was removed under reduced pressure and redissolvedin chloroform and this mixture washed extensively with 1% NaCl to removeunreacted SMPB and residual triethylamine. The product of this reactionwas characterized by two dimensional TLC and proton NMR. This analysisindicated the presence of two components, one being MPB-PE andcomprising approximately 60% of the product. The product of the reactionmixture described above was incorporated into vesicles withoutadditional Purification. The product was stored at -20° C. and was shownto be stable for at least 6 months.

II. Synthesis of N-[3(2-Pyridyldithio)Proprionyl]phosphatidylethanolamine (PDP-PE)

PDP-PE was prepared according to the procedure of Leserman et al.,Liposome Technology, III. Gregoriadis, ed., 1984, CRC Press, Inc., CA.,p. 29-40. Briefly, 50 umol EPE was dissolved in 3.5 mlchloroform/methanol (9:1) and added to 1.5 ml methanol containing 60umol SPDP and 100 umol triethylamine. After a four hour incubation atroom temperature, TLC (solvent: chloroform/methanol/water, 65:25:4)analysis indicated 99% conversion of EPE to a faster running product.This reaction mixture was washed with 10 ml of phosphate buffered saline(0.1M NaCl, 0.1M potassium phosphate, pH 7.4). This was repeated threeadditional times prior to removal of the organic phase under reducedpressure. Further analysis by two dimensional TLC and proton NMRindicated a single product which appeared greater than 98% pure. PDP-PEwas stored under N₂ in chloroform at -20° C. and was stable for at leastsix months.

III. Vesicle Preparation

Large unilamellar vesicles (LUVs) were prepared employing the LUVET(LUV's by extrusion techniques) procedure described by Hope et al.,Biochim, Biophys. Acta, 812, p. 55-65 (1985). Appropriate mixtures oflipid were deposited as a dry lipid film by evaporation from chloroformunder a stream of nitrogen gas, placed under vacuum for at least twohours and subsequently hydrated with appropriate buffer by vortex mixingto produce multilamellar vesicles (MLVs). Frozen and thawed MLV (FATMLV)systems as described in Mayer et.al, Biochim. et Biophys. Acta, 817,193-196 (1985), were obtained by freezing the MLV's in liquid nitrogenand thawing at 40° C., a cycle which was repeated five times. TheseFATMLV's were then extruded under nitrogen pressure ten times throughtwo stacked Nucleopore polycarbonate filters of defined pore size.Generally a pore size of 1OO nm was employed resulting in a preparationof unilamellar vesicles (VET₁₀₀) with an average diameter of 11O nm asdetermined by QELS measurements. Vesicles of variable sizes wereproduced by similar extrusion techniques through filters with pore sizesranging from 30 nm to 400 nm. Unless specified differently vesicles wereprepared in a NaCl/EPPS buffer (150 mM NaCl, 20 mM EPPS,(N-2-Hydroxyethylpiperazinepropanesulphonic acid) pH 8.0) at a finallipid concentration of 10 umol/ml. In vesicles which contained PDP-PE,the presence of this lipid was verified by measuring the release of2-thiopyridinone upon addition of DTT (25 mM final concentration) asindicated previously.

IV. Preparation of Proteins Used for Coupling

IgGs and streptavidin were modified with the amine reactiveheterobifunctional reagent SPDP according to Leserman et al. (supra.).Biotin-conjugated antibodies were prepared according to Bayer et al.,Biochim et Biophys Acta, 550, p. 464-473, (1979).

V. Covalent Coupling of Proteins to Vesicles

The steps utilized to crosslink proteins to liposomes are as follows:thio-reactive lipids were generated, PDP-PE and MPB-PE, according to theprocedures of Leserman, et al., supra., and Martin et al., supra.,respectively. These derivatized PE's were incorporated into vesicles, atlevels of 5 mol % based on total modified lipid content. Since theMPB-PE was shown to be less than 60% pure by two dimensional TLC and ¹H-NMR, the vesicles prepared with "MPB-PE" actually contained only 3% ofthis modified lipid. Thus, for vesicles prepared as described above, 100ul of vesicles were added to 100 to 500 ul of the protein solutiondepending on protein concentration. This reaction mixture (pH=8.0) wasincubated in the dark under nitrogen for 12 to 18 hours unless specifieddifferently. Subsequently the liposomes were separated from unassociatedprotein employing a Sepharose 4B-CL column (column volume equivalent toa least 20 times the sample volume) equilibrated with NaCl/Hepes buffer.Protein and total lipid concentrations in the fractions were determineddirectly or calculated from the specific activities of ¹²⁵ I-labeledprotein and [³ H]-DPPC, respectively. Where FITC-labeled proteins wereused fractions were dissolved in ethanol to a total volume of 2 ml andthe fluorescence was determined and compared to the fluorescenceassociated with a known quantity of fluoresceinated protein. Thepresence of lipid was shown not to influence fluorescence in this assaysystem.

Binding of biotinated-IgGs to avidin-coupled vesicles was assessed asdescribed above after a 30 minute incubation at room temperature. IgGwas generally added at equimolar concentrations with respect to avidinpresent in the incubation mixture.

Protein modification with the heterobifunctional reagent SPDP was shownto be extremely reproducible provided the DPDP was freshly prepared andstored anhydrously. This reaction resulted in the substitution for IgGin the range of 4 to 5 moles 2-dithiopryidinone (2-DT) per mole IgG(regardless of the IgG used) and for protein A and streptavidin 6 to 7moles 2-DT per mole protein based on an incubation ratio of 10 molesSPDP per mole protein. It was demonstrated that this extent ofmodification did not influence the binding activity of a monoclonal IgG(anti-transferring receptor IgG) or the ability of streptavidin to bindbiotin.

The reaction mixture used for coupling consisted of vesicles containingone of the thio-reactive lipids plus the modified protein bearingseveral thiol groups, as judged by the release of 2-DT on addition ofDTT. Following an incubation period of 18 hours, the vesicles andassociated protein were fractionated from free protein on Sepharose4B-CL columns and the lipid and protein were quantified as indicated inthe methods.

Martin et al. (Ann. N.Y. Acad. Sci., 446, p. 443-456, 1985) hadsuggested that the thio-reactive PE derivatives function better in thepresence of cholesterol. The amount of IgG coupling obtained withvesicles prepared with PDP-PE or MPB-PE was compared as a function ofcholesterol content. The results, shown in FIG. 1, indicate that IgGcrosslinking to vesicles containing PDP-PE does not occur unless thecomposition includes greater than 20 mol % cholesterol. Conversely,levels of 12 ug IgG per umole lipid were obtained for vesiclescontaining the SMPB derivative of PE even in the absence of cholesterol.This amount of association increased linearly with respect to the amountof cholesterol included in the vesicles to levels approaching 30 ug IgGper umole lipid for vesicles containing 45 mol % cholesterol.

The reaction of SPDP modified proteins with MPB-PE was extensivelycharacterized. The time course for coupling IgG (FIG. 2A) suggests thatthe reaction proceeds in two phases. Approximately 25 ug of IgG wascoupled to vesicles within one hour. Subsequently, the rate ofassociation decreased several fold, but continued linearly for at least18 hours. As indicated in FIG. 2B, maximum coupling was obtained whenthe vesicles were prepared with 5 mol % MPB-PE. Similar levels of IgGcoupling were obtained with 10 mol % MPB-PE, however there was asignificant amount of vehicle crosslinking in this preparation as judgedby an increases in the optical density of the reaction mixture andgreater than 50% loss of both lipid and protein on the Sepharose 4B-CLgel filtration column. The amount of IgG coupling to vesicles increasedlinearly with increased amounts of modified IgG present in theincubation mixture (FIG. 2C). Assuming a vesicle diameter of 100 nm,levels of 170 ug IgG per umole lipid, also resulted in vesiclecrosslinking.

The extent of protein coupling was shown to be dependent on the pH ofthe reaction mixture (FIG. 3). Contrary to previous work which performedsimilar coupling reactions at pH 6.7, (Heath et.al, Proc. Natl. Acad.Sci U.S.A., 80, p. 1377-1381, (1983), Bragman et al., Biochim. Biophys.Acta, 730, p. 187-195, (1983), and Bragman et al., JNCI, 73, p. 127-131,(1984), modified IgG and streptavidin coupling to vesicles occurred onlyat pHs greater than 8.0. At pH values below 7.0, only backgroundprotein-lipid association was observed.

As shown in FIG. 4, the amount of IgG crosslinking was dependent on thesize of the vesicle employed. The maximum efficiency of the couplingreaction never exceeded 45% based on the amount of protein present inthe reaction mixture. However, for vesicles which had been sized throughthe 200 nm pore size filters, showing an average diameter of 162+/-41 nmby QELS measurements, the ratio of available MPB-PE to IgG approached 1indicating that the maximum level of crosslinking was obtained. Based onthe QELS approximated average diameters, 3950, 75, and 20 IgG moleculeswere bound to the 162 nm, 115 nm and 82 nm vesicles, respectively.Similar results have been obtained for sonicated vesicles when comparedto vesicles produced by the reverse phase-evaporation technique andsized through 0.2 and 0.4 um pore size filters.

Finally, the influence of lipid composition on the coupling of proteinsto vesicles containing MPB-PE was determined. The results shown in Table1 emphasize previous results (FIG. 1), and demonstrate that IgG couplingto vesicles is much more efficient when cholesterol is incorporated.Crosslinking of the smaller streptavidin (68,000 daltons vs. 150,000daltons for IgG) was not influenced by addition of this sterol. Thesedata also indicate that incorporation of either a negative charge(phosphatidylserine or "PS") or a positive charge (stearylamine or "SA")into the liposome does not influence the extent of coupling for eitherprotein.

VI. Binding of biotinated-IgG to stepavidin coupled vesicles("Avisomes")

An advantage can be obtained if a single vesicle preparation is employedto couple a variety of different immunoglobulins. It has been shown thatnot all IgGs can be modified with the reagent SPDP such that they retainbinding activity (Heath et.al, supra), and that certain IgGs which havebeen extensively modified with SPDP could not be crosslinked efficientlyby the procedures described above. Leserman et. al, supra., recognizedthese limitations and circumvented them somewhat by coupling protein Ato liposomes. Since protein A binds the Fc portion of IgGs of certainsubclass (IgG2a), protein A coupled vesicles specifically bound to cellspreincubated with a variety of antibodies. This allowed for a comparisonof a number of different parameters using a single vesicle preparation.A similar yet more general approach can be achieved by taking advantageof the strong affinity (Kd=10⁻¹⁵ M) of biotin for streptavidin. SinceIgGs can be easily biotinated, Bayer et al., supra; and Heitzmann et al.Proc. Natl. Acad. Sci. U.S.A., 71, p. 3537-3541, (1974), a streptavidincoupled vesicle can be used to bind any number of IgGs with differingspecificities. This application of "Avisomes" is illustrated in FIG. 5,which shows the elution profile for "avisomes") following a thirtyminute incubation with biotinated-IgG (4 biotins per IgG). More than 40ug IgG were bound per umole lipid which corresponded to approximately100 IgGs per vesicle. Similar levels of association were obtained forthree different IgG and could be obtained at biotin/IgG ratios of lessthan 2. In addition, the efficiency of this association was better thanthat observed for the chemical crosslinking procedures employedpreviously.

VII. Dehydration of protein-coupled vesicles and entrapment ofadriamycin

Strepavidin-coupled vesicles ("avisomes") were dehydrated according tothe procedure of Madden et al., Biochim. Biophys Acta, 817, p. 67-74,(1985). Vesicles were prepared as described above in a citrate buffer(100 mM citric acid, 150 mM KOH, pH 4.5) which contained 250 mMtrehalose. Subsequently the external buffer was exchanged for NaCl/EPPSbuffer, thereby establishing a transmembrane electrical potential whichcan be utilized for accumulating adriamycin. Following the previouslydescribed coupling reaction, unassociated protein was separated fromvesicles as described above employing a Sepharose 4B-CL columnequilibrated with NaCl/Hepes buffer containing 250 mM trehalose.Vesicles with bound protein were divided into 1 ml aliquots and dried in10 ml Kimex tubes at room temperature under vacuum for 24 hours.

Following dehydration, samples were rehydrated by addition of 900 uldistilled water. The resulting preparation was characterized withrespect to binding of several biotinated-IgG and the ability toaccumulate adriamycin. Adriamycin was quantitated by determining theabsorbance at 480 nm of a triton X-100 solubilized sample which has beenfractionated on Sephadex G-50 to remove unassociated adriamycin.

As shown in FIG. 6 "avisomes" can accumulate adriamycin in response to apreexisting pH gradient, where levels of 180 and 120 nmoles adriamycinper umole lipid are obtained for streptavidin coupled vesicles composedof EPC and EPC/Chol (1:1), respectively.

The ability to store "avisomes" in a dehydrated form was thendemonstrated (Table 2). In these experiments vesicles were prepared inthe presence of trehalose with removal of untrapped trehalose performedPrior to coupling, by gel filtration. Following the coupling reactiontrehalose was added (final concentration =250 mM) to the streptavidincoupled vesicles and then dehydrated as described previously. There waslittle change in the amount of streptavidin bound to rehydrated"avisomes". Moreover, the biotin binding activity of streptavidinassociated with vesicles (units per umole lipid) was not influenced bythis dehydration step. This is also reflected in the ability of thesepreparations to bind biotinated-IgG to the same extent observed prior todehydration.

EXAMPLE 2 Non-Covalent Coupling

Biotinylated PE was incorporated into egg phosphatidylcholine (EPC) at amolar ratio of 0.1% (with respect to PC) and LUVs produced by anextrusion procedure through 100 nm filters (Hope et al., supra.), andusing a freeze and thaw technique (Bally et al., 1985, Biochim. etBiophys. Acta., 812, 66-76), resulting in vesicles of approximately 100nm diameter. The vesicles were incubated at 25° C. for 30 minutes at pH8.0 in 10 fold molar excess of streptavidin (with respect to PE) in 20mM EPPS buffered saline. At 5, 10, 15 and 20 minute intervals, aliquotswere fractioned on Sepharose C14B columns (5 ml) to separateliposomally-bound streptavidin from free streptavidin (FIG. 7). Theratio of streptavidin bound per umole total lipid was determined to beat streptavidin:biotinylated PE ratios of 1:12 (mol/mol). This resultedin a maximum of 5.8 ug of streptavidin bound per umol lipid.

Biotinylated anti-rat erythrocyte IgG was prepared with 1-5 biotinscovalently bound per mole of antibody by the method of Bayer et al.,supra. FITC-labelling of biotinated antibodies was performed byincubation of antibody (5 mg/ml in PBC) with celite-FITC (2.5 mg/ml in0.1 mM NaCl, 0.2M Na bicarbonate, pH 8.8) for 20 minutes at 25C,followed by gel filtration on Sepharose G50 (50 ml column).Antibody-streptavidin-liposomes were prepared by incubation ofFITC-labelled biotin antibody (1 mg/ml) with streptavidin liposomes(1-2.5 umoles/ml) for 30 minutes at 25° C., at a 4-fold mole ratio ofantibody to streptavidin. The final product was separated from freeantibody by gel filtration on a Sepharose C14B column (15 ml).Phospholipid was assayed using the standard phosphate assay of Bartlettet al.)

EXAMPLE 3 Non-Covalent Coupling

The methods of Example 2 were followed using 0.05, 0.15, 0.25, 0.35, and0.5 mole % biotinylated PE with EPC in the liposomes. This linearlyincreased the amount of streptavidin bound per liposome, by increasingthe number of sites available for biotinylated antibody to couple toliposomes. A constant ratio of streptavidin was maintained with respectto total lipid.

EXAMPLE 4 Targeting of Non-Covalently Coupled Systems

Biotinylated anti-rat IgG or Biotinylated F(ab)2 fragments were coupledto LUVs as in Example 2. These LUVs contained 125I-labelled tyramineinsulin (25 nmoles EPC, 25 nmoles biotin PE, 0.027 uCi 125I insulin/umollipid), according to the procedures of Sommerman et al., 1984, Biochim.et Biophys. Res Comm., 122, 319-324. The high specific activity of thisentrapped marker allowed the in vitro distribution of the vesicleantibody complexes to be determined. For the preparation of biotinatedF(ab)₂ fragments, biotinated anti-rat erythrocyte IgG (4 biotins/IgG)was digested with pepsin in 0.1M Na acetate, pH 4.5 at 37° C. overnight(Nisoff et al., 1960, Arch. Biochem. Biophys., 89, 230-244). Theproducts were fractionated on a Sephadex G150 column and fractionscontaining F(ab)₂ fragments, as determined by 10% SDS polyacrylamide gel(Laemmli, 1970, Nature. 227, 680-685) were pooled. F(ab)₂ or antibodystreptavidin liposomes were prepared as in Example 2. For erythrocytecell binding studies, rat or human erythrocytes were washed with 20 mMEPPS buffered saline, pH 8, three times. Lipid (0.62 umol/ml) wasincubated with 10⁹ erythrocytes in each experiment for 1 hour at 4° C.,with the exception of (a) (see Table 1), where the lipid concentrationwas 1.76 umol/ml. Cells were washed three times with 20 mM EPPS bufferedsaline, PH 8, and were counted to determine levels oferythrocyte-associated liposomes.

As shown in Table 3, little non-specific binding of biotinylatedliposomes to rat or human erythrocytes was observed. Anti-raterythrocyte IgG or F(ab)₂ liposome complexes bind specifically to raterythrocytes but not to human erythrocytes.

                  TABLE 1                                                         ______________________________________                                        EFFECT OF LIPID COMPOSITION ON COUPLING                                                    ug IgG per   ug Strepavidin per                                  Lipid Composition                                                                          umole Lipid  umole Lipid                                         ______________________________________                                        DPPC/CHOL    41           N.D.                                                DPPC         8.2          N.D.                                                DOPC/CHOL    42           38.3                                                DOPC         20           36.0                                                EPC/CHOL     27           42                                                  EPC          4.3          36                                                  ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        STORAGE OF STREPAVIDIN COUPLED VESICLES                                                  ug Strepavidin per                                                                          Units per                                                                              Biotinated                                             umole Lipid   umole    igG per                                     Sample     (ug from units)                                                                             Lipid    umole Lipid                                 ______________________________________                                        24 Hrs in Buffer                                                                         41            N.D.     N.D.                                        24 Hrs in  43(56)        0.80     42.75                                       Trehalose                                                                     Dehydrated with                                                                          50 (57)       0.80     42.65                                       Trehalose                                                                     ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Binding of targeted liposomes to rat erythrocytes                                                No.                                                                           of liposomes bound per                                                          rat       human                                          Sample               erythrocyte                                                                             erythrocyte                                    ______________________________________                                        Liposomes            18        ND                                             Streptavidin-liposomes                                                                             20        ND                                             .sup.(a) Pre-incubation with IgG +                                                                 542       ND                                             streptavidin-liposomes                                                        IgG streptavidin-liposomes                                                                         416       11                                             IgG streptavidin-liposomes + biotin                                                                73        ND                                             F(ab)2 streptavidin liposomes                                                                      302       11                                             F(ab)2 streptavidin liposomes + biotin                                                             50        ND                                             ______________________________________                                         ND = Not Done                                                            

We claim:
 1. A streptavidin-coupled liposome.
 2. Thestreptavidin-coupled liposome of claim 1 wherein the streptavidin iscoupled to the liposome covalently.
 3. The streptavidin-coupled liposomeof claim 1 wherein the streptavidin is coupled to the liposomenon-covalently.
 4. The streptavidin-coupled liposome of claim 1 whereinthe liposome contains a bioactive agent.
 5. The streptavidin-coupledliposome of claim 1 wherein the liposome has a transmembrane potential.6. The streptavidin-coupled liposome of claim 5 wherein the liposomecontains a bioactive agent.
 7. The streptavidin-coupled liposome ofclaim 1 wherein the composition is dehydrated.
 8. Thestreptavidin-coupled liposome of claim 1 wherein the liposome contains abioactive agent and has a transmembrane potential, and wherein thecomposition is dehydrated.
 9. A pharmaceutical composition comprisingthe streptavidin-coupled liposome of claim 1 and a pharmaceuticallyacceptable carrier or diluent.
 10. The streptavidin-coupled liposome ofclaim 1 wherein streptavidin is additionally coupled to a biotinatedprotein.
 11. The streptavidin-coupled liposome of claim 10 wherein theprotein is Immunoglobulin G.
 12. The streptavidin-coupled liposome ofclaim 10 wherein the protein is a monoclonal antibody.
 13. A method forpreparing a liposome non-covalently coupled to streptavidin comprisingthe steps of:(a) preparing liposomes comprising biotinylatedphosphatidylethanolamine; and (b) incubating the liposomes instreptavidin.
 14. The method of claim 13 wherein the liposomes are largeunilamellar vesicles.
 15. The method of claim 13 wherein the liposomesalso comprise egg phosphatidylcholine.
 16. The method of claim 15wherein the biotinylated phosphatidylethanolamine is in an about0.1%-0.5% mole ratio with the egg phosphatidylcholine.
 17. The method ofclaim 16 wherein the biotinylated phosphatidylethanolamine is in anabout 0.1% mole ratio with the egg phosphatidylcholine.
 18. The methodof claim 13 wherein step (b) is performed in about 10 fold molar excessof streptavidin to biotinylated phosphatidylethanolamine.
 19. A methodfor preparing a protein-coupled streptavidin-biotinylatedphosphatidylethanolamine-containing liposome wherein streptavidin isnon-covalently coupled to the liposome, comprising the steps of:(a)forming the streptavidin-coupled biotinylated phosphatidylethanolamineliposome of claim 13, and (b) incubating a biotinylatedfluorescent-amine-labelled protein with the liposomes.
 20. A compositioncomprising a protein coupled to streptavidin which is non-covalentlycoupled to a liposome comprising biotinylated phosphatidylethanolamine.21. The composition of claim 20 wherein the liposome is a largeunilamellar vesicle.
 22. The composition of claim 20 wherein the proteinis an immunoglobulin or a monoclonal antibody.
 23. The composition ofclaim 22 wherein the immunoglobulin is Immunoglobulin G.
 24. Thecomposition of claim 20 additionally comprising a bioactive agent.
 25. Apharmaceutical composition comprising the composition of claim 20 and asuitable pharmaceutical carrier or diluent.